Stable cytochrome P450 24 (CYP24) expressing cell line and methods and uses thereof

ABSTRACT

The invention relates to a stable recombinant host cell comprising a CYP24 nucleic acid. The host cell is preferably a mammalian cell or an insect cell. The cell is useful for identifying modulators of CYP24 that can be used in the treatment of CYP24 expression-related medical conditions.

RELATED APPLICATION

This application claims the benefit of U.S. provisional patentapplication No. 60/376,806, filed May 2, 2002, entitled “CYP24 StableCell Line For Drug Screening”.

FIELD OF THE INVENTION

The invention relates to a stable cell line expressing the cytochromeP450 24 (“CYP24”) and methods and uses thereof. It also relates to arecombinant cell that expresses CYP24.

BACKGROUND OF THE INVENTION

The cytochrome P450 s comprise a large gene superfamily that encodesover 500 distinct heme-thiolate proteins that catalyze the oxidation ofdrugs and numerous other compounds in the body. It is of considerableinterest in the pharmaceutical and other fields to identify cytochromeP450 s and the role they play in the metabolism of individual compounds.Cytochrome P450 s are heme—containing enzymes that strongly absorb at awavelength of 450 nm when the heme is bound to a molecule of carbonmonoxide. They are most well known for their ability to catalyze themetabolism of a wide variety of drugs, xenobiotics, carcinogens,mutagens and pesticides, and they are also involved in catalyzingreactions that make or degrade cholesterol, steroids, and other lipids.The reactions performed by these enzymes are generally oxidations,hydroxylations, acetylations, and demethylations. Mutations incytochrome P450 s or abnormal expression levels can cause a number ofhuman diseases such as glaucoma and breast cancer. Cytochrome P450 s arealso involved in the metabolism of a number of vitamins, such as VitaminA (retinoic acid) [White et. al. (1996) J. Biol. Chem. November, 22:271(47): 29922-7; WO/97/49815; WO 01/44443] and Vitamin D [Jones, G. et.al. (1999) July; 140(7):3303-10; Dilworth F J, et. al. (1995) Jul. 14;270(28); 16766-74. In particular, cytochrome P450 s, CYP27 and CYP24,are involved in Vitamin D₃ and D₂ metabolism. Vitamin D₃ and D₂, bothseco-steroids, are metabolized into their active forms by CYP27 and arethen further metabolized by CYP24. CYP24 is a mitochondrial cytochromeP450. that has previously been characterized. For example, isolatedhuman CYP24 was published in Chen et al. (Isolation and expression ofhuman 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad SciUSA 1993 May 15; 90(10):4543-7). In Chen et al. it was reported that thehuman 24-hydroxylase 1539 base pair open reading frame encoded a 513amino acid sequence, 90% homologous to rat CYP24. Mouse CYP24 wascharacterized in Yoshimura et al. (Molecular cloning of25-hydroxyvitamin D-3 24-hydroxylase (Cyp-24) from mouse kidney: itsinducibility by vitamin D-3. Biochim Biophys Acta 1995 Oct. 17;1264(1):26-8).

The vitamin D metabolic pathway is part of a vital endocrine system thatis highly regulated at certain stages and produces metabolites thatcontrol the secretion of the parathyroid gland hormones. 1α,25(OH)₂D₃, ahormone produced in the vitamin D pathway, regulates phosphate andcalcium levels in the blood which in turn control bone mass, the stateof bones, and affect cellular differentiation in the skin and the immunesystem. In the vitamin D pathway, cytochrome P450 s introduce functionalgroups by hydroxylation usually at positions 1, 25, and 24 of thesteroid.

The metabolism of vitamin D begins with 25-hydroxlyation of vitamin D₃or D₂ in the liver to 25(OH)D₃. 25(OH)D₃ and a second metabolite,1α,25(OH)₂D₃, are converted to 24,25(OH)₂D₃ and 1,24,25(OH)₃D₃ by CYP24,a mitochondrial P450 involved in the vitamin D pathway. CYP24 is inducedby 1,25(OH)₂D₃ and is found in the kidney as well as other vitamin Dtarget tissues such as the parathyroid cells, keratinocytes,osteoblasts, and enteroctyes.

There are a number Vitamin D related medical conditions. Moreinformation on Vitamin D conditions can be found in the Proceedings ofthe Workshop on Vitamin D (Walter de Gruyter publishing, Berlin),proceedings 1 to 11. For instance, vitamin D deficiency has been relatedto the following:

-   -   (i) in the parathyroid—hyper- and hypo-parathyroidism,        Osudohypo-parathyroidism, Secondary hyperparathyroidism;    -   (ii) in the pancreas—diabetes;    -   (iii) in the thyroid—medullary carcinoma;    -   (iv) in the skin—psoriasis;    -   (v) in the lung—sarcoidosis and tuberculosis;    -   (vi) in the kidney—chronic renal disease, glomerulonephritis,        IgA nephropathy, membraneous nephropathy, glomerulosclerosis,        nephrosis, renal insufficiency, hypophosphtatemic VDRR, vitamin        D dependent rickets;    -   (vii) in the bone—anticonvulsant treatment, fibrogenisis        imperfecta ossium, osteitits fibrosa cystica, osteomalacia,        hypocicemia, osteporosis, osteopenia, osteosclerosis, renal        osteodytrophy, rickets;    -   (viii) in the intestine—glucocorticoid antagonism, idopathic        hypercalcemia, malabsorption syndrome, steatorrhea, tropical        sprue;    -   (ix) in the prostate—cancer; and    -   (x) in the breast—cancer.

More common conditions related to vitamin D or vitamin D metabolitedeficiency are obesity problems, hyperhoshatemic turmoral calcinosis,sarcoidosis, tuberculosis, primary hyperparathyroidism, vitamin Ddependent rickets type II, cholestatic or paremchymal liver disease.

Excess levels of Vitamin D can be toxic and can cause conditions such ashyperglycemia and mental deficiency. Such conditions usually presentthemselves upon excess ingestion of Vitamin D.

Since CYP24 is involved in maintaining Vitamin D homeostasis and isimplicated in the development of these diseases, it is important tounderstand how CYP24 activity is and can be modulated in vivo and invitro. For this, there is a need for a cellular model system that stablyexpresses CYP24. Such a model system would be especially useful in invitro drug development studies. To date, there has been no stable cellline expressing CYP24. Cell lines that only generate transientexpression are unsuitable for drug development assays. Some cell linesmay also not be able to produce active protein (i.e. protein that isfolded properly and able to perform a function, the function of nativeCYP24 such as catalysis). In the absence of a stable cell line, onecannot attribute reduced CYP24 activity to the effects of a candidateinhibitor when it is also possible that the cells merely have lost theirability to express CYP24. There is also a need for a cell model systemthat not only expresses CYP24 but expresses an active form of thepeptide (e.g. that can fold properly and has endogenous CYP24activities). There is also a need for a cell model system that allowsrecombinant CYP24 to be active within the cell.

SUMMARY OF THE INVENTION

The invention provides a stable cell line expressing the cytochrome P450CYP24. The invention also relates to cells and cell cultures of saidcell line. The invention further relates to methods and uses of the celllines.

In one embodiment, the invention provides a cell line that stablyexpresses CYP24, comprising a recombinant CYP24 nucleic acid moleculethat is operably linked to a promoter to enable expression thereof,preferably in biologically active form thereof. In a further embodimentthe cells enable CYP24 expression without exogenous cofactors.

In another embodiment the CYP 24 nucleic acid molecule is a nucleic acidmolecule that codes for a CYP24 polypeptide. In another embodiment theCYP24 nucleic acid molecule is selected from the group consisting ofHPK-1A ras cell CYP24 [SEQ ID NO:1], or one of Genbank Accession Nos.U60669, NT011362, XM030593, AK016668, NM009996, AF312914, NM000782,AL138805, AF245504, AF126400, AF149309, X59506, AH002273, L04619, L4618,L04617, L04616, L04615, L04614 and L04613, Q07973, Q64441, Q09128,NP₀₃₄₁₂₆, AAA42340, U60669, AH002273 or any DNA sequence encoding anamino acid sequence corresponding to one of the foregoing sequences. Inone embodiment, the CYP24 nucleic acid molecule has at least 70%sequence identity to [SEQ. ID. NO. 1], more preferably 90% sequenceidentity to [SEQ. ID. NO. 1]. In yet another embodiment, the nucleicacid molecule is a homolog, analog, derivative or obvious chemicalequivalent, degenerate sequence or mutation that still encodes for CYP24polypeptide.

In one embodiment the cells of the cell line are mammalian, such as V79hamster cells, or insect cells, such as SF9 cells.

In another embodiment, the recombinant CYP24 nucleic acid molecule isfrom a vector comprising CYP24 nucleic acid molecule. For example, saidvector can be a plasmid, cosmid, a viral or retroviral vector andselected for optimization with the host cell. In one embodiment, thevector is suitable for transfecting Sf9 cells, such as a baculovirusvector, such as pFastBac1. In another embodiment, the vector is suitablefor transforming or transfecting a mammalian cell, such as pcDNA3.1. Inone embodiment the vectors further comprise an antibiotic resistancegene, such as neomyocin (neo+) or hygromycin (hygro+) or other markersuitable for selection of transfected or transformed cells.

In one embodiment, the vector is pFBCYP24H6, pFBCYP24 orpcDNA3.1-CYP24H6 or pcDNA3.1-CYP24. In another embodiment the pcDNA3.1is neo+ or hygro+.

In another embodiment, the cell line is derived from Sf9 cellstransfected with pFBCYP24H6 or pFBCYP24, preferably pFBCYP24H6. In afurther embodiment, the cell line is derived from V79 hamster cellstransformed with pcDNA3.1-CYP24 or pcDNA3.1-CYP24H6, more preferablypcDNA3.1-CYP24.

In another embodiment, the invention provides a method of making a cellline of the invention expressing CYP24 comprising:

-   -   (i) isolating a suspension of cells,    -   (ii) inserting in the cells a recombinant gene encoding CYP24 so        that it is operably linked to a promoter and a selectable        marker, such as, but not limited to an antibiotic resistant        gene, to produce a culture containing cells that comprise said        recombinant gene, and    -   (iii) enriching the culture by selectably growing cells        expressing CYP24 in media differentially favorable for growth of        the cells expressing CYP24, to obtain a cell line expressing        CYP24.

The invention includes a cell culture comprising cells of the invention.The cells stably express CYP24 and include a recombinant CYP24 nucleicacid molecule that is operably linked to a promoter to enable expressionthereof, in a medium capable of sustaining growth and replication of thecells.

In another embodiment, the invention provides a method of identifying amodulator of a CYP24 polypeptide comprising,

-   -   (i) culturing the cell line that stably expressed CYP24 of the        invention under conditions wherein the cell expresses the        polypeptide CYP24 in the presence of a CYP24 substrate and a        candidate modulator; and    -   (ii) determining whether the candidate modulator modulates CYP24        substrate activity, wherein increased or decreased CYP24        activity indicates that the candidate modulator is a modulator        of the CYP24 polypeptide.

In an embodiment, the CYP24 activity is monitored by one or more of thefollowing:

-   -   a. monitoring binding of the candidate modulator with the        substrate;    -   b. monitoring of CYP24 induced substrate metabolites;    -   c. monitoring binding of CYP24 with the candidate modulator;        and/or    -   d. monitoring CYP24 gene expression.

In a further embodiment of the method for identifying the modulator, theeffect of the candidate modulator is determined by comparing the affectof said candidate modulator with that of a control. For instance, thecontrol can comprise conducting the method in the absence of thecandidate modulator or in the presence of a positive control such asketoconazole, a known CYP24 inhibitor.

In the method of identifying the modulator, in an embodiment, the stepof determining whether the candidate compound modulates CYP24polypeptide activity comprises adding a substrate to the cell anddetecting increased or decreased activity of the CYP24 on the substratein the presence of the candidate compound.

In another embodiment, the cell line can be used for generating CYP24comprising culturing said cell line under conditions that enable and/orpromote CYP24 expression. In a further embodiment the cells of theinvention are cultured under conditions that enable CYP24 activity. Amethod of isolating CYP24 comprising expressing CYP24 in a cell line ofthe invention and isolating the expressed CYP24 is also herein provided.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be described in relation to thedrawings in which:

FIG. 1 RT-PCR analysis of single-cell cloned CYP24/V79 cells.

FIG. 2 Levels of CYP24 expression in isolated mitochondria.

FIG. 3 Effect of co-factors on CYP24 activity.

FIG. 4 CYP24 activity in single-cell cloned CYP24/V79 cells.

FIG. 5 Stability of CYP24/V79 cells in culture (one month).

FIG. 6 Effect of incubation time on CYP24 activity.

FIG. 7 Effect of incubation time on CYP24 activity.

FIG. 8 1,25(OH)₂D3 metabolism by CYP24/V79 cells.

FIG. 9 Experimental controls of Example 6, which include wellscontaining dead cells (DC-10 μM) and wells without cells (NC-10 μM and 1nM). V79-4, V79-CYP24 and HPK1A-ras cells were cultured.

FIG. 10A Metabolism of 10 μM 1α,25-(OH)₂D₃: organic and aqueous phases.

FIG. 10B Metabolism of 500 nM 1α,25-(OH)₂D₃: organic and aqueous phases.

FIG. 11 Inhibition of Ketoconazole (CYP24 stable transfected celllines).

FIG. 12A Nucleic acid coding region sequence [SEQ ID NO:1] (1560bp)—amplified region of CYP24 for stable cell line construct. Thesequence for CYP24 in pcDNA3.1-hygro(+) is the coding sequence of CYP24with GCTAGCACC at 5′ end (the first six nucleotides are for NheI site[SEQ. ID. NO. 9]) before ATG and CTCGAG [SEQ. ID. NO:10] after stopcodon TAA at 3′ end (the sequence for XhoI site). The sequencecorresponds to Genebank Accession no. (NCBI) XM_(—)030593.

FIG. 12B. Amino acid sequence [SEQ ID NO:2]—gi|14786394|ref|XP_(—)030593.1| cytochrome P450, subfamily XXIVprecursor [Homo sapiens].

FIG. 13. Full length homo sapiens nucleic acid sequence CYP24 (3274 bp)[SEQ ID NO:3]>gi|147863931|ref|XM_(—)030593.1| Homo sapiens cytochromeP450, subfamily XXIV (vitamin D 24-hydroxylase) (CYP24), mRNA.

FIG. 14. Graph showing inhibition of CYP24 activity by candidatecompounds.

DETAILED DESCRIPTION OF THE INVENTION

The invention satisfies the need for a stable cell line expressing thecytochrome P450 CYP24.

“Cell line” as used herein means a population or mixture of cells ofcommon origin growing together after several passages in vitro. Bygrowing together in the same medium and culture conditions, the cells ofthe cell line share the characteristics of generally similar growthrates, temperature, gas phase, nutritional and surface requirements. Thepresence of cells in the cell line expressing CYP24 can be ascertained,provided a sufficient proportion, if not all, of cells in the line arepresent to produce a measurable quantity of the substance. An enrichedcell line is one in which cells having certain trait e.g. expression ofCYP24, are present in greater proportion after one or more subculturesteps than the original cell line. Preferably the cell line is derivedfrom one, two or three originating cells. The cell line can become morehomogenous with successive passages and selection for specific traits.Clonal cells are those which are descended from a single cell.

“Recombinant cell” as used herein means a cell that has been geneticallymodified from its native form by way of transfection with a foreign(non-endogenous) genetic (nucleic acid) material.

A cell line or cell comprising a recombinant CYP24 nucleic acid moleculeis a cell line or cell that comprises a CYP24 nucleic acid molecule thatis not endogenous to said cell line or cell, as the case may be. Thecell line or cell preferably produces no or low endogenous CYP24expression. The cell line is stable in that it expresses CYP24 for morethan a transient period of time. The stable cell line should expressCYP24 for a minimum of at least one month, and preferably at least twomonths, at least four months and most preferably at least 6 months or 12months. The CYP24 produced by the cells is biologically active in thatmetabolizes 25(OH)D₃ and 1α,25(OH)₂D₃, to 24,25(OH)₂D₃ and1,24,25(OH)₃D₃. The cell lines of the invention are preferablyimmortalized. The cells are optionally subcultured periodically, such asabout once every three days. The cells are also optionally passaged atleast 20 times, at least 40 times or at least 100 times. Certaindiseases cause mammals to express significantly altered levels of CYP24protein and mRNA levels encoding CYP24 protein when compared to acorresponding “standard” mammal i.e., a mammal of the same species nothaving the condition. Cancer would be one example of where CYP24 levelsare increased. In other conditions, CYP24 levels may be decreased orinsufficient.

The stable cell lines of the invention are very useful to identifymodulators to inhibit or activate CYP24 to treat these conditions. SinceCYP24 expression is consistent for a prolonged period of time in thestable cell line of the invention, a researcher can reliably attributemodulation of CYP24 activity to the activating or inhibiting effects ofa candidate compound.

“Regulatory elements” or “Regulatory sequences” as used herein meansthose elements or sequences in addition to those directly coding for theamino acid sequence in question that regulate the expression of theamino acid sequence in question. Such elements or sequences include, butare not necessarily limited to: promoters, operons or other sequencesthat need to be operably linked to the CYP24 nucleic acid molecule toenable expression or regulation of expression of CYP24.

“Modulator” as used herein means any substance (e.g. drug, chemical,peptide, antibody, nucleic acid molecule) or condition (temperature,salt levels, pH, etc.) that can increase, decrease or maintain (e.g.homeostasis—increase or decrease as required) CYP24 expression oractivity. These can include any agonist, antagonist or simulator.

“CYP24 peptide, polypeptide or protein” is an amino acid sequence from afamily of cytochrome P450's that catalyses the following reaction:Vitamin D metabolites—25(OH)D₃ and 1α,25(OH)₂D3, to 24,25(OH)₂D₃ and1,24,25(OH)₃D₃, respectively. As used herein, “CYP24” or “CYP24peptide”, “CYP24 polypeptide” or “CYP24 protein” are usedinterchangeably.” has the amino acid sequence as shown in SEQ. ID. NO. 2or that of a homolog, a species homolog, analog, or derivative of SEQ.ID. NO. 2 that has the above-noted enzymatic activity. “CYP24” alsoincludes a biologically active fragment or obvious chemical equivalentof SEQ. ID. NO. 2, homolog, species homolog, analog or derivativethereof.

CYP24 polypeptide may include various structural forms of the primaryprotein that retain biological activity. For example, a polypeptide ofthe invention may be in the form of acidic or basic salts or in neutralform. The CYP24 polypeptides may be modified by either naturalprocesses, such as post-translational processing or by chemicalmodification techniques, which are well known in the art. Suchmodifications are described in basic texts, research manuals andresearch literature. Modifications may occur anywhere in the CYP24including the peptide backbone, the amino acid side-chain and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given CYP24 polypeptide. In addition, a given CYP24 maycontain many types of modification. The modifications may result frompost-translational natural processes or may be made by syntheticmethods.

The term “analog” includes any polypeptide such as CYP24 having an aminoacid residue sequence substantially identical to the CYP24 sequencesdescribed in this application in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays CYP24 activity as described herein. Examples of conservativesubstitutions include the substitution of one non-polar (hydrophobic)residue such as alanine, isoleucine, valine, leucine or methionine foranother, the substitution of one polar (hydrophilic) residue for anothersuch as between arginine and lysine, between glutamine and asparagine,between glycine and serine, the substitution of one basic residue suchas lysine, arginine or histidine for another, or the substitution of oneacidic residue, such as aspartic acid or glutamic acid for another. Thephrase “conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue provided thatsuch polypeptide displays the requisite activity.

The term “derivative” refers to a polypeptide such as CYP24 derivativehaving one or more residues chemically derivatized by reaction of afunctional side group. Such derivatized molecules include for example,those molecules in which free amino groups have been derivatized to formamine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5 hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

“Obvious chemical equivalent” as used herein means a compound (e.g.nucleic acid molecule, peptide, or other compound) or a method of makingstable CYP24 cells or using stable CYP24 cells that has no materialeffect on the way that the invention works. The fact that the varianthas no material effect will be obvious to a reader skilled in the art.Examples of obvious chemical equivalents include but are not limited toobvious variations of CYP24, degenerate CYP24 nucleic acid sequences,vectors or reagents and conservative amino acid substrates of CYP24.

“Sequences having substantial sequence homology” means those polypeptideor nucleic acid sequences which have slight or inconsequential sequencevariations from [SEQ ID NOS:1-3] or one of the other sequences describedin this application, i.e., the sequences function in substantially thesame manner and, with respect to nucleic molecules code for functionallyequivalent proteins. The variations may be attributable to localmutations or structural modifications.

The peptides of the invention can be isolated from cells expressing saidpolypeptide using techniques known in the art. In another embodiment,the peptides of the invention can be synthetically made using techniquesknown in the art.

CYP24 Nucleic Acids

CYP24 nucleic acid is a family of nucleic acid cytochrome P450's thatincludes many types of CYP24 nucleic acids. As used herein, a CYP24nucleic acid is a nucleic acid molecule that codes for CYP24 polypeptideas defined above. A preferred CYP24 nucleic acid is the isolated cDNAsequence obtained by RT-PCT of the CYP4 mRNA from the HPK-1A ras cellCYP24 [SEQ ID NO:1] (FIG. 12A). The corresponding amino acid sequence isshown as [SEQ ID NO:2] (FIG. 12B) and the full length nucleic acidsequence is shown as [SEQ ID NO:3] (FIG. 13) CYP24 may be obtained fromother sources, such as the sequences in Genbank Accession Nos. U60669,NT011362, XM030593, AK016668, NM009996, AF312914, NM000782, AL138805,AF245504, AF126400, AF149309, X59506, AH002273, L04619, L4618, L04617,L04616, L04615, L04614, L04613, Q07973, Q64441, Q09128, NP034126,AAA42340, U60669 or AH002273. Any DNA sequence encoding an amino acidsequence corresponding to one of the foregoing sequences would beuseful. Any nucleic acid sequence encoding a polypeptide correspondingto one of the above-encoded sequences would be useful.

Thus, a CYP24 “nucleic acid” refers to isolated nucleic acids whichencode CYP24 clone peptides and to obvious chemical equivalents thereof,including degenerate nucleic acid sequences.

A CYP24 “nucleic acid” also refers to isolated nucleic acids whichencode the amino acid sequence in [SEQ ID NO:1 or 3], or a biochemicallyactive fragment thereof.

CYP24 “nucleic acid” also includes polynucleotides comprising nucleicacid sequences having substantial sequence homology with the sequencesof SEQ. ID. NOS. 1 or 3. Preferably, nucleic acids are capable ofhybridizing, under stringent hybridization conditions, to [SEQ ID NO:1or 3], or the complement thereof. “Stringent hybridization conditions”refers to an overnight incubation at 42° C. in a solution comprising 50%formamide, 5×SSC [750 mM NaCl, 75 mm sodium citrate], 50 mM sodiumphosphate [pH 7.6], 5× Denhardt's solution, 10% dextran sulfate, and 20ug/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C.

In addition, CYP24 nucleic acids may contain one or more modified basesor DNA or RNA backbones modified for stability or for other reasons.“Modified” bases include, for example, tritiated bases and unusual basessuch as inosine. A variety of modifications can be made to DNA and RNA;thus, “nucleic acid” embraces chemically, enzymatically, ormetabolically modified forms.

CYP24 nucleic acids also include variants having at least: 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to [SEQ ID NO:1 or3] that code for a CYP24 polypeptide. Sequence identity can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between [SEQ ID NO:1 or 3]a subject sequence, also referred to as a global alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al., Comp. App. Biosci. 6: 237-245 (1990). In a sequencealignment, the query and the subject sequence are both DNA sequences. AnRNA sequence can be compared by converting U's to T's. The result ofsaid global sequence alignment is in percent identity. Preferredparameters used in a FASTDB alignment of DNA sequences to calculatepercent identity are: Matrix-Unitary, k-tuple=4, Mismatch Penalty=1,Joining Penalty=30, Randomization Group Length=09, Cutoff Score=1, GapPenalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of thesubject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the result. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specific parameters, toarrive at a final percent identity score. This corrected score is whatis used for the purposes of the present invention. Only bases outsidethe 5′ and 3′ bases of the subject sequence, as displayed by the FASTDBalignment, which are not matched/aligned with the query sequence, arecalculated from the purposes of manually adjusting the percent identityscore.

Also provided in the present invention are homologs of CYP24, such asspecies homologs. Species homologs may be isolated and identified bymaking suitable probes or primers from the sequences provided herein andscreening a suitable nucleic acid source for the desired homolog.

CYP24 nucleic acids and polypeptides can be obtained using techniquesknown in the art. Such methods include isolating naturally occurringpolypeptides and polynucleotides, recombinantly orsynthetically/chemically produced polynucleotides or polypeptides or acombination of these methods.

An isolated CYP24 nucleic acid molecule which comprises DNA can beisolated by preparing a labeled nucleic acid probe based on all or partof the nucleic acid sequences as shown in [SEQ. ID. NOS. 1 or 3] andusing this labeled nucleic acid probe to screen an appropriate DNAlibrary (e.g. a cDNA or genomic DNA library). For example, a genomiclibrary isolated can be used to isolate a DNA encoding a CYP24 proteinby screening the library with the labeled probe using standardtechniques. Nucleic acids isolated by screening of a cDNA or genomic DNAlibrary can be sequenced by standard techniques.

An isolated CYP24 nucleic acid molecule, which is DNA, can also beisolated by selectively amplifying a nucleic acid encoding a CYP24protein using the polymerase chain reaction (PCR) methods and cDNA orgenomic DNA. It is possible to design synthetic oligonucleotide primersfrom the nucleic acid sequence as shown in [SEQ. ID. NOS. 1 or 3] foruse in PCR. A nucleic acid can be amplified from cDNA or genomic DNAusing these oligonucleotide primers and standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.

A CYP24 isolated nucleic acid molecule, which is RNA, can be isolated bycloning a cDNA encoding a novel protein of the invention into anappropriate vector, which allows for transcription of the cDNA toproduce an RNA molecule, which encodes a protein of the invention. Forexample, a cDNA can be cloned downstream of a bacteriophage promoter,(e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro withT7 polymerase, and the resultant RNA can be isolated by standardtechniques.

A CYP24 nucleic acid molecule may also be chemically synthesized usingstandard techniques (eg, Wendell McKenzie. DNA Synthesis. (1994, Gordonand Breach Publishing Group, USA)). Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis, which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

Determination of whether a particular nucleic acid molecule encodesCYP24 may be accomplished by expressing the cDNA in an appropriate hostcell by standard techniques, and testing the activity of the proteinusing the methods as described herein. A cDNA having the activity of aCYP24 protein so isolated can be sequenced by standard techniques, suchas dideoxynucleotide chain termination or Maxam-Gilbert chemicalsequencing or by automated DNA sequencing, to determine the nucleic acidsequence and the predicted amino acid sequence of the encoded protein.

An “anti-sense CYP24 nucleic acid” as used herein is a nucleotidesequence that is complementary to a target, preferably CYP24 mRNA orDNA. In another embodiment the antisense sequence targets part of themRNA or DNA encoding CYP24. Preferably, an antisense sequence isconstructed by inverting a region preceding or targeting the initiationcodon or an unconserved region. In particular, the nucleic acidsequences contained in the nucleic acid molecules of the invention or afragment thereof, preferably a nucleic acid sequence shown in [SEQ. ID.NOS. 1 or 3] may be inverted relative to their normal presentations fortranscription to produce antisense nucleic acid molecules. In oneembodiment the antisense molecules can be used to inhibit CYP24expression.

Stable CYP24 Cells

CYP24 nucleic acid molecules may be incorporated into an appropriate,commercially available expression vector according to procedures knownin the art in order to ensure good expression of the protein in a hostcell. Such vectors may be viral, retroviral, plasmids, and cosmids. Itis also known in the art that the selection of a suitable vector willalso depend on the host cell selected and vise versa. It is known in theart that certain vectors are more suitable with certain host cells. Forinstance, it is known that baculovirus vectors, such as pFastBac1 workwell in insect cell lines, such as Sf9 and plasmids such as pcDNA3.1,pcDNA 3.1-Hygro(+) work well in mammalian cells, such as V79 hamstercells. One example uses the commercially available plasmid pFastBac1 tomake pFB-CYP24H6 or pFBCYP24. These were then used to transfect sF9cells to get an SF9 CYP24 and CYP24H6 expressing stable cell line(baculoviruses expressing CYP24 and CYP24H6). For Sf9 cells, the amountof virus used relative to the number of cells and the infection timewill affect the expression level of CYP24. In another embodiment,pcDNA3.1-CYP24 and pcDNA3.1-CYP24H6 were made and used to transform V79cells, to create V79 CYP24 and CYP24H6 expressing stable cell line.Another vector used was pBS-H6 to make pBSCYP24H6. The inventionincludes a recombinant expression vector containing CYP24 nucleic acidand the necessary regulatory sequences for production of CYP24 in astable cell line. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, or viral genes (forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Selection of appropriate regulatory sequences isdependent on the host cell chosen, and may be readily accomplished byone of ordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector. It will also be appreciated that the necessary regulatorysequences may be supplied by the native protein and/or its flankingregions. Suitable methods for transforming and transfecting host cellscan be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other suchlaboratory textbooks.

Mammalian and insect cells are preferred host cells. V79 hamster cell isan example of a suitable mammalian cell. Sf9 is an example of a suitableinsect cell. Both V79 and Sf9 cells are readily available fromcommercial cell suppliers, such as the ATCC and other tissue culturecollections. Other suitable host cells are readily apparent andavailable to those skilled in the art. Suitable cells for transfectionpreferably have no or low endogenous levels of CYP24 to enable one tomonitor the affect of certain factors (including candidate chemicalmodulators), on CYP24 expression and/or activity. Preferred cell linesexpress the cofactors necessary for CYP24 activity such as adrenodoxin(ADX) and adrenodoxin reductase (ADR).

“Conditions suitable for CYP24 expression” as used herein means theculture conditions, be it media, the presence of substrate or cofactors,pH, temperature or other environmental conditions that are required forCYP24 expression.

In one embodiment, the expressed CYP24 incorporates into themitochondrial membrane of the host cell and the addition of exogenouscofactors such as NADPH, ADR and/or ADX is not required for CYP24activity. In another embodiment, exogenous NADPH, ADX and ADR are addedto the cells to enhance CYP24 activity. Recombinant CYP24 in active formis also produced without addition of cofactors (endogenous cofactors inthe cell are used).

In another embodiment, the cell lines of the present invention can be asource of CYP24. CYP24 can be isolated therefrom using techniques knownin the art.

CYP24 Modulators

This invention further provides methods for screening compounds toidentify modulators, e.g. activators and inhibitors of CYP24 and itsvariants. Inhibitors include antibodies, drugs, small molecules,peptides, or in some cases, oligonucleotides. An activator is a compoundthat enhances the function or increases the levels of polypeptide.Inhibitors block the function or decrease the levels of polypeptide.Modulators also include agonsists, antagonists of CYP24 expressionand/or activity.

CYP24 activity can be directly inhibited by the binding of a smallmolecule or drug. The present invention thus includes a method ofscreening drugs, chemicals, compounds (novel and/or known compounds) fortheir effect on activity and/or expression (i.e., as a modulator) ofCYP24 polypeptide. In particular, modulators of CYP24 activity, such asdrugs or peptides, can be identified in a biological assay by expressingCYP24 in a cell, adding a substrate and detecting activity of CYP24polypeptide on the substrate in the presence or absence of a modulator.In other instances, the substrate is added to induce CYP24 expression inthe presence or absence of a candidate modulator. Thus, the CYP24protein can be exposed to a candidate compound and the effect on proteinactivity can be determined. Prospective drugs can be tested formodulation of the activity of other P450 cytochromes, which are desirednot to be modulated. In this way, drugs that selectively modulate CYP24over other P450 s can be identified.

The effect on CYP24 expression and/or activity can be determined usingtechniques known in the art. For instance, affect on CYP24 expressioncan be monitored by monitoring the presence and/or levels of CYP24 mRNA.The effect on CYP24 activity can be monitored by monitoring the presenceand/or levels of substrate metabolite(s). This can be monitored inreference to controls. The controls can be positive or negativecontrols. It could be external and/or internal controls. For instance,activity and/or expression levels of a candidate modulator can beascertained by comparing said levels with an assay done in the presenceof a known modulator (activator or inhibitor, as the case may be), inthe absence of a modulator (on the same cells or parallel cell lines),in cells that endogenously express CYP24 (e.g. Human ras K cells).Binding of a candidate modulator with the substrate in thepresence/absence of CYP24 also indicate potential modulating activity.Similarly binding of the candidate modulator with CYP24 in the presenceof the substrate, is also indicative of modulating activity. Suchbinding assays are well known in the art. Potential modulators of CYP24activity are CYP24 cofactors or combinations thereof such as theaddition of both ADX and ADR enhances CYP24 expression in the cell lineof the present invention.

A potential inhibitor is a peptide derivative of CYP24 (e.g. a naturallyor synthetically modified analog) that has lost catalytic function yetstill recognizes CYP24 substrates. Examples of peptide derivativesinclude, but are not limited to, small peptides or peptide-likemolecules. An inhibitor could also be a Vitamin D analog or derivativethat competes with Vitamin D for binding with CYP24.

Antisense constructs prepared using antisense technology are alsopotential inhibitors. Therefore, the present invention is furtherdirected to inhibiting polypeptide in vivo by the use of antisensetechnology. [for example, antisense-Okano, J. Neurochem. 56: 560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CPRPress, Boca Raton, Fla. (1988)]. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription[triple-helix, see Lee et al., Nucl. Acids Res. 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991)], thereby preventing transcription and the production of theCYP24 polypeptides.

The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

Antibodies to CYP24 and or substrate may also inhibit CYP24 activity.

Modulators may also be designed using CYP24 peptide and/or substratestructure information.

Pharmaceutical Compositions

There are diseases or conditions in which a subject is administered apharmaceutical composition comprising an effective amount of a compoundthat inhibits CYP24 expression or activity. An inhibitor identifiedusing the stable cell line is optionally administered in combinationwith 1,25 vitamin D3. The inhibitor will allow the active vitamin D toexert its effect.

The pharmaceutical compositions for administration to subjects areformulated in a biologically compatible composition suitable foradministration in vivo. As used herein “biologically compatible formsuitable for administration in vivo” means a form of the substance to beadministered in which therapeutic effects outweigh any toxic effects.The substances may be administered to animals in need thereof.

The pharmaceutical composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (and potential side effects), thesite of delivery, the method of administration, the scheduling ofadministration, and other factors known to practitioner. Administrationof a “therapeutically effective amount” of pharmaceutical compositionsof the present invention is defined as an amount of the pharmaceuticalcomposition, at dosages and for periods of time necessary to achieve thedesired result. For example, a therapeutically active amount of asubstance may vary according to factors such as disease state, age, sex,and weight of the recipient, and the ability of the substance to elicita desired response in the recipient. Dosages may be adjusted to providean optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. Subject totherapeutic discretion, preferably dosages of administration of activecompound will be in the range of about 1 μg/kg/day to 10 mg/kg/day ofpatient body weight and most preferably at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day.

An active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, topical, intratumoral etc.),oral administration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activesubstance may be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound, prior to reaching the desired site of delivery.It can also be formulated into a sustained release composition.

The compositions described herein can be prepared by known methods forthe preparation of pharmaceutically acceptable compositions which can beadministered to subjects, such that an effective quantity of the activesubstance is combined in a mixture with a pharmaceutically acceptablecarrier. Suitable carriers are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA 1985). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

Recombinant nucleic acid molecules comprising a sense, an antisensesequence or oligonucleotide fragment thereof, may be directly introducedinto cells or tissues in vivo using delivery vehicles known in the artsuch as retroviral vectors, adenoviral vectors and DNA virus vectors.They may also be introduced into cells in vivo using physical techniquesknown in the art such as microinjection and electroporation or chemicalmethods such as coprecipitation and incorporation of DNA into liposomes.Recombinant molecules may also be delivered in the form of an aerosol orby lavage.

EXAMPLES

The following non-limiting examples are illustrative of the presentinvention:

Example 1 Generation of Expression Vectors with Full-length CYP24 cDNA

HPK-1A ras cells were treated with 10 nM 1,25(OH)₂D3 for 18 hours. Cellswere collected and total RNA was prepared using Trizol reagent. Thefull-length CYP24 cDNA was amplified using one-step RT-PCR kit(Clontech) according to the protocol suggested by the supplier. Theforward primer used is 5′-TACGCTAGCACCATGAGCTCCCCCATCAGCAA-3′ (SEQ. ID.NO. 4) and the reverse primer used is5′-AGGCTCGAGTTATCGCTGGCAAAACGCGATGG-3′ (SEQ. ID. NO. 5) An NheI site andan XhoI site (underlined) were engineered in the forward and the reverseprimer before the start codon and after the stop codon, respectively.The 1570 bp PCR fragment was digested with NheI and XhoI and the 1560 bpNheI-XhoI fragment was cloned into the mammalian expression vectorpcDNA3.1-Hygro(+) (Invitrogen) that had been digested with the sameenzymes. The ligation was used to transform E. coli TOP 10 cells(Invitrogen) and the transformed cells were incubated at 30° C.overnight. The recombinant pcDNA3.1-CYP24 was screened and the fidelityof the PCR fragment was confirmed by sequencing.

To generate the his-tagged version of CYP24, the C-terminal portion ofthe gene was amplified by PCR using the full-length cDNA cloned inpcDNA3.1 as template. The forward primer used is5′-CCCAAAGGAACAGTGCTCATGC-3′ (nt 1228-1249) (SEQ. ID. NO. 6) and thereverse primer used is 5′-TGGCTCGAGTCGCTGGCAAAACGCGATGGGG-3′ (SEQ. ID.NO. 7) The reverse primer removes the stop codon and has an XhoI site(underlined). The 320 bp PCR fragment was digested with BamHI and XhoIand the 280 bp BamHI-XhoI fragment was cloned into the tagging vectorpBS-H6 that had been digested with BamHI and SalI. The fidelity of thePCR fragment was confirmed by sequencing. This resulted in the additionof 8 amino acids, VDHHHHHH [SEQ. ID. NO. 8], at the C-terminus of CYP24.Then the resulting vector was digested with BamHI and XhoI and the 300bp BamHI-XhoI fragment was recovered and ligated to the backbone ofpcDNA3.1-CYP24 that had been digested with the same enzymes.Subsequently, the his-tagged full-length cDNA was excised and clonedinto the donor plasmid pFastBac1 to give pFB-CYP24H6.

Example 2 Expression of CYP24 in Sf9 Insect and V79 Hamster Cells

The recombinant donor plasmid, pFB-CYP24H6, was used to generatebaculovirus encoding his-tagged CYP24, according to the suggestions ofthe supplier (Gibco Life Technology). The expression of his-tagged CYP24was confirmed by immunoblotting on whole cell lysate. The virus wasexpanded and titration determined.

To generate a stable V79 cell line for CYP24, pcDNA3.1-CYP24 wasmaxi-prepared and used to transfect V79 hamster cells using FuGENEreagent (Roche) as recommended by the supplier. The transfected cellswere selected for two weeks in the presence of 100 μg/ml hygromycinbefore being single-cell cloned into 96-well plate (seed 50 cells perplate) to isolate the expression clones. Thirty populations of cellsderived from a single cell in the 96-well plate were expanded into6-well plate. Total RNA was later isolated from 24 expanded clones usingTrizol reagent. RT-PCR analysis was performed using one-step RT-PCR kit(Clontech) and 13 positive clones (# 2, 3, 9, 10, 11, 13, 14, 16, 17,21, 22, 23, and 26) were expanded and stocks prepared.

Example 3 Requirements of Co-factors for His-tagged CYP24 Expressed inInsect Sf9 Cells

To examine the effect of co-factors on the activity of CYP24,recombinant baculoviruses encoding his-tagged adrenodoxin (ADX),adrenodoxin reductase (ADR), or both were generated. His-tagged CYP24was expressed in insect cells either alone, or with adrenodoxin,adrenodoxin reductase, or both. Three hundred ml of Sf9 cells (0.8million cells/ml) in TNM-FH medium (BD Pharmingen) were cultured inroller bottles in the presence of 2 μg/ml hemin chloride, 100 μMδ-amino-levulinic acid, and 100 μM ferric citrate. Baculovirusesencoding his-tagged CYP24 and co-factors were added at multiplicity ofinfection of 2. The cells were collected 66-70 hours after infection andthe mitochondrial fraction was prepared as described below.

Preparation of Mitochondrial Fraction

In one example, cells are placed in cold MT buffer (100 mM potassiumphosphate buffer, pH 7.5, and 0.25 M sucrose) and transferred to 50 mltubes. The cells are centrifuged at 800 g for 10 min at 4° C. and thesupernatant is discarded. Protease inhibiting tablet is dissolved in MTbuffer (one tablet/10 ml of MT buffer). CaCl₂ is added to 0.25 mM andthe mix is vortexed to suspend cells in the buffer, with about 10 ml per150 million Sf9 cells. Cells are transferred into the chamber ofpre-cooled nitrogen bomb. The cells are equilibrated for 10 minutesunder pressure of 300 psi. Cells are slowly released from the bomb intothe 50 ml tube. Repeat the nitrogen bomb steps. EGTA is added to 1 mMfinal. The mix is centrifuged at 800 g for 10 minutes at 4° C. Thesupernatant is combined in a Beckman 50 ml tube for JA20 rotor. Thepellet is washed once with 10 ml MT buffer and the supernatants combinedtogether followed by centrifugation at 10,000 g for 10 minutes at 4° C.The supernatant is discarded and the pellet washed once with 20 ml of MTbuffer followed by centrifugation again at 10,000 g for 10 min at 4° C.The supernatant is discarded. The tube is drained by inverting the tubeon paper tower for a minute. One ml of MT buffer is added to the tube.The mitochondrial fraction (pellet) is homogenized. The proteinconcentration of the mitochondrial fraction is determined. Samples arediluted to 5 mg/ml. Immunoblotting is done with 10 μl of sample andstore the rest in cryogenic vials at −70° C. freezer until needed. CYP24expressing mitochondria retain full function after being stored at −70°C. for three months.

The expression levels of his-tagged CYP24 in isolated mitochondria weredetermined by immunoblotting. Mitochondrial protein was diluted to 0.2μg/μl with MT buffer and an equal amount of 2 times SDS loading bufferwas added. Samples were mixed and heated at 100° C. for 5 min. Ten μl ofeach sample was loaded on 12.5% SDS-PAGE. Separated proteins weretransferred to polyvinylidene fluoride (PVDF) membrane and expression ofhis-tagged CYP24 and co-factors was detected using anti-his antibody(FIG. 2). The results showed that CYP24 was expressed at comparablelevels under these conditions.

Primary antibody: monoclonal Penta-His antibody (QIAGEN) used at 0.1ng/ml.

Secondary antibody: HRP conjugated goat anti mouse Ig used at 1:10,000.

CYP24 activity in these four samples was determined. Mitochondrialfraction prepared from Sf9 cells infected with a control virus was alsoincluded. Two hundred μg of mitochondrial protein was used in each assayin 200 μl in the presence of 100 mM potassium phosphate, pH 7.5, 250 mMsucrose, 1 mM DTT, 1 mM EDTA, 5 mM MgCl₂, 1 mM NADPH, 5 mM D,L-trisodium isocitrate, and 0.2 units of isocitrate dehydrogenase. Thereaction was started by adding 50,000 CPM of [³H-1β]-1,25(OH)₂D3 in 1 μlof ethanol. The reaction was carried out at 37° C. for 15 min. Then thereaction was extracted using Bligh-Dyer extraction method (see below).The radioactivity present in the aqueous phase was counted and thepercentage of conversion was calculated as following:Percentage of conversion=[(total aqueous CPM in sample tube)−(totalaqueous CPM in control tube)]*100%/(total CPM added)

The Bligh-Dyer extraction method is preferred. An example of volumes ofreagents is provided for 200 μl of reaction. First, 5 μl of 10% aceticacid and 0.5 ml of methanol are added to each tube (well).Dichloromethane (0.25 ml) is added to each tube and vortexed for 30 s.Next, 0.25 ml of dichloromethane is added followed by 0.25 ml ofsaturated KCl. The mix is spun at 4,000 rpm for 10 min. Next, 100 μl ofthe aqueous phase (top, total 950 μl) is used for radioactivity countingand the rest of the aqueous phase is aspirated. The organic phase(bottom) is vacuum dried then d dissolved in 150 μl mobile phase andspun at 10,000 rpm for 5 min. Next, 140 μl of sample is transferred tothe insert for HPLC analysis.

The results showed that mitochondria from Sf9 cells could support thefunction of CYP24 (FIG. 3). The presence of adrenodoxin reductase had nosignificant effect on CYP24 activity, while co-expression of adrenodoxinsomehow suppressed CYP24 activity. However, in the presence of bothco-factors, CYP24 activity increased 4-5 fold.

Example 4 Screening of Stable CYP24/V79 Transfectants with HighEnzymatic Activity

Cells of the 13 RT-PCR positive CYP24/V79 clones were further screenedby CYP24 activity assay. Cells were seeded in duplicate in 6-well plate(for initial screening, cell number is not standardized). No cell andV79 cell controls were also included in the assay. After 18 hours, 1 μlof [³H-1β]-1,25(OH)₂D3 (50,000 CPM/μl) in ethanol was added to each welland incubated for 24 hours. Cells were then extracted with Bligh-Dyermethod. The radioactivity present in the aqueous phase was counted. Thepercentage of conversion for each clone was calculated as following:Percentage of conversion=[(total CPM in sample well)−(total CPM in nocell control well)]*100%/(total CPM added)

FIG. 4 showed that CYP24 activity in clone No. 10, 13, and 21 wasrelatively low. The rest of the ten clones were maintained in culturefor up to 3 month and CYP24 activity was checked every month. This time500,000 cells of each clone were seeded in 6-well plate. The results inFIG. 5 showed that after one-month incubation, only clone No. 14 stillhas high CYP24 activity. The activity remains the same after 3 months.

Example 5 Functional Characterization of CYP24 Expressed in Hamster V79Cells

CYP24 activity in clone No. 14 was further characterized. First, a timecourse was carried out. CYP24/V79 cells were suspended, counted, and500,000 cells were seeded in 6-well plate in 0.6 ml of medium containing1.5 nM [³H-1β]-1,25(OH)₂D3. Cells were extracted at 0, 3, 6, 12, and 24hours using Bligh-Dyer extraction method. The radioactivity present inaqueous phase was compared. FIG. 6 revealed that after 3 hours, theradioactivity in the aqueous is almost saturated. So another time coursewith shorter incubation time was carried out. Cells were extracted at 0,0.5, 1, 2, and 3 hours and radioactivity present in aqueous phasecompared. FIG. 7 showed that CYP24 activity in clone No. 14 is linear upto 3 hours. The organic phase from 3 hours was analyzed by HPLC and theresults in FIG. 8 showed that the substrate was disappeared and a numberof metabolites could be seen.

Example 6 HPLC Based Metabolic Comparison of CYP24 Expressing CellSystems

Cell culture. V79-4, V79-CYP24 and HPK1A-ras cells were cultured in 150mm tissue culture dishes with 25 ml of Dulbecco's Modified Eagle Medium(DMEM) supplemented with 10% (v/v) fetal bovive serum (FBS) and 5% (v/v)antibiotic/antimycotic in a humidified incubator at 37° C./5% CO₂. Themedia used for the V79-CYP24 cells was also supplemented with 0.4% (v/v)hygromycin B. subcultured into 6 well plates. After recovery, only theHPK1A-ras containing wells were treated with 10 nM 1α,25-(OH)₂D₃ for 18hours.

Incubation with 1α,25-(OH)₂D₃. At approximately 80% confluence, V79-4,V79-CYP24 and HPK1A-ras cells were subcultured into 6 well plates. Afterrecovery, only the HPK1A-ras containing wells were treated with 10 nM1α,25-(OH)₂D₃ for 18 hours. At incubation time, the media was removedand the cells were rinsed with 2 ml of phosphate buffered saline (PBS).2 ml of bovine serum albumin supplemented media (1% (w/v)) containingthe appropriate amount of 1α,25-(OH)₂D₃ was introduced to the wells.Each well was supplemented with 2 μl of 100 mM 1,2-dianilinoethane(DPPD) antioxidant. Each cell line was incubated with 10 μM, 1 μM, 500nM and 1 nM (150 000 CPM) 1α,25-(OH)₂D₃ or [1β-³H] 1α,25-(OH)₂D₃ for 24hours in triplicate wells. Experimental controls included wellscontaining dead cells (DC-10 μM) and wells without cells (NC-10 μM and 1nM) according to the scheme in FIG. 9.

Total Lipid Extraction and High Performance Liquid Chromatgraphy. 1 μgof 1α—OH-D₃ was added to each well as an internal recovery standard.Media and cells were extracted according to a method modified from thatof Bligh and Dyer (Bligh, E., Dyer, W. Can. J. Biochem. 37: 911-917.1959). The aqueous phase was re-extracted with 5 ml methylene chloridein the presence of 0.01% (v/v) glacial acetic acid in order to isolatethe water soluble catabolites. The organic phase was subjected to normalphase HPLC using a solvent system of 91/7/2-hexane/isopropanol/methanol(percentage of total flow) at a flow rate of 1 ml/minute on Zorbax-SIL(3μ) [Agilent]. The aqueous phase re-extract was subjected to reversedphase HPLC using an acetonitrile/water based gradient system over 30minutes in the presence of 0.1% (percentage of total flow) glacialacetic acid. A flow rate of 1 ml/minute was used on Zorbax SB-C18 (3.5μ)[Agilent]. Metabolite quantitation was facilitated by diode arraydetection (UV₂₆₅). Metabolite identification was based on observation ofthe characteristic vitamin D chromophore (λ_(max)=²⁶⁵; ε=18 300) and onco-chromatography with authentic synthesized standards. On-lineradioflow chromatography of the samples incubated with [1β-³H]1α,25-(OH)₂D₃ also facilitated metabolite identity, and subsequent MSanalyses are expected to confirm these observations.

The stable transfected cell line V79-CYP24 was shown to possess similarcapacity to metabolize 1α,25-(OH)₂D₃ via the intermediates ofC24-oxidation to calcitroic acid when compared to a previouslywell-studied CYP24 expressing system, the human keratinocyte cell line,HPK1A-ras. Metabolism was assessed based on substrate disappearance andtotal product formation. Conversely, the un-transfected V79-4 cellsshowed no metabolism. Substrate concentration dependent patterns ofmetabolite production are conserved between the two systems, such thatat high concentrations (10 μM) the proximal pathway intermediatespredominated, whereas at the low concentrations, the more polarmetabolites were the most prevalent (1-500 nM) (FIGS. 10A and 10B)(Kaufmann, M., Masuda. S., and Jones, G. 2001. 23^(rd) Annual Meeting ofthe American Society for Bone and Mineral Research, Phoenix, Ariz., USA.Oct. 12-16, 2001. J. Bone Miner. Res. 16:Supp. 1. Abstract SA529. PosterPresentation. These observations confirm that the metabolites previouslyobserved in other vitamin D target cell systems, are due to CYP24catalyzed catabolism.

Example 7 Identification of Modulators of CYP24

Ketoconazole(cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxyl]phenyl]piperazine)was administered to CYP24/V79 cells. CYP24/V79 cells were suspended,counted, seeded on well plates and extracted using the Bligh-Dyerextraction method. The organic phase from 3 hours was analyzed by HPLCand the results showed that activity of CYP24 decreased (FIG. 11). Sinceketoconazole reduced CYP24 activity, these results show thatketoconazole is an inhibitor of the polypeptide. The protocol used inthe ketoconazole experiment is shown below. This protocol can be used toscreen for other modulators of CYP24.

Other candidate compounds were screened using the same method and areused to identify both activators and inhibitors of CYP24. Otherinhibitors, for example, include erythromycin and itraconazole. Theactivators and inhibitors are useful in vitro and in vivo.

Example 8

CYP24 Enzyme Assay Using Stable CYP24-V79 Cells—(Inhibition byKetoconazole or Modulation by Other Test Compounds)

CYP24-V79 cells and HPK1A-ras cells were subcultured. For example, 2million CYP24-V79 cells were optionally subcultured in 1* 150 mm platetwo days before the assay. HPK1A-ras cells were preferably induced, forexample, 18 hours prior to the start of the assay. To induce cells,media was removed and the cells washed with 1× PBS buffer. About 16-18mL of fresh media was added to each large plate and 16-18 ml of 10⁻⁵ M1,25(OH)₂D₃ (i.e. 1 mL of 10⁻⁵ M 1,25(OH)₂D₃ per 1 mL media (10⁻⁸ Mfinal concentration)).

Next, a cell suspension was prepared at the day of assay by removing themedium and washing the cell with PBS. 2 ml tripsin/EDTA was added to theplate which was kept in 37° C. for 5 min. Next, 3 ml EDTA medium+1% FCSwas added followed by transfer to a 50 ml tube. The plate was washedwith 5 ml PBS and mixed. The mix was centrifuged (2,000 rpm, 5 min) andthe suspended cells pelleted in DMEM medium+1% BSA.

In one embodiment, since the V79-CYP24 cells lift quickly they were heldat 37° C. for about 2 minutes but the HPK1A-ras cells were left forapproximately 10 minutes. About 5 mL 1× PBS buffer was added to collectcells from the plate and then placed in a 50 mL tube followed byrepetition with second cell line. About 5 mL 1× PBS buffer was added tofurther wash the plate and added to the original 50 mL tube. Tubes werecentrifuged (2,000 rpm/800 g for 6 minutes) and the supernatant removed.The pellet was resuspended in DMEM+1% BSA media (initial resuspensionvolume will depend on assay requirements, i.e. if require 12 mL forassay, start there). The cells were counted via hemocytometer anddensity adjusted to 250,000 cells/150 μL (1.67 million/1 mL) and then150 μL added to appropriately labeled wells of 48-well plate. Cells hadabout 30 minutes in the incubator to adhere to the wells. The cells werecounted and cell density was adjusted to 500,000/150 μL. Then the 150 μLcell suspension was added to each well in a 48-well plate (including 3well as no cell control (NCC)), and a 3 well cells without drug orinhibitor as control). For example:

No No No NCC NCC NCC T T T K-8 K-8 K-8 K-7 K-7 K-7 K-6 K-6 K-6 K-5 K-5K-5

About 25 μL medium was added containing ketoconazole (examples of finalconcentrations include 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M or 5×10⁻⁷, 5×10⁻⁸,or 5×10⁻⁹). The IC₅₀ for ketoconazole is around 5*10⁻⁷ M. The plate waskept in 37° C. for 10 min.

Other test compounds (inhibitor or activator) were used at a series ofvariable concentrations such as 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰ or5×10⁻⁷, 5×10⁻⁸, or 5×10⁻⁹.

Substrate was prepared with a predetermined amount of DMEM+1% BSA medium(25*well number+200) μL in a tube, followed by adding an amount of³H-1,25(OH)₂D₃ (well number+2) μL and a certain amount of 100 mM DPPD(well number/5) μL and mixing them by vortex.

DMEM + 1% BSA 550 μL DPPD  4.4 μL ³H-1 α, 25(OH)₂D₃ (50,000 CPM/μL)  11μL mix

To incubate the samples, 25 μL substrate was added to each well and theplate was incubated at 37° C. for 2 hour. 25 μL substrate was added tothe counting plate (2 wells) as a total count.

In the assay, wells (optionally eight wells) remained untreated withinhibitor to provide controls. Substrate was added to four of thesewells (100% cell control). To the remaining four wells, nothing wasadded until after the reaction was stopped with the first addition ofmethanol. At this point, 25 mL of the substrate solution (0% cellcontrol) was added. Some substrate solution was set aside, and 25 mLadded directly to scintillation fluid when counting.

Lipid extraction (Bligh-Dyer) and counting were done by adding 500 μLmethanol to each well to stop the reaction, followed by transfer to atube. Next, 250 μL dichloromethane was added followed by vortexing. Anadditional 250 μL dichloromethane was added along with 250 μL saturatedKCl. The mix was centrifuged at 4000 rpm for 5 min. About 100 μL ofaqueous phase (upper phase) was transferred to a plastic counting platewith 600 μL of scintillation fluid was added to each well. The plate iscounted in a scintillation counter. Enzyme activity was then calculated.CPM of cell control after subtraction of CPM of NCC is as 100% enzymeactivity.

References for the above protocol include:

-   Ray S, Ray R, Holick M. Metabolism of ³H-1alpha, 25-dihydroxyvitamin    D₃ in the cultured human keratinocytes (1995) 59:117-122-   Dilworth F J, Scott I, Green A, Strugnell S, Guo Y D, Roberts E A,    Kremer R, Calverley, M J, Makin H L J, Jones G. Different mechanisms    of hydroxylation site selection by liver and kidney cytochrome P450    species (CYP27 and CYP24) involved in Vitamin D metabolism. (1995) J    Biochem 270(28):16766-16774

In another example, Vitamin D analog candidate inhibitor compounds,called I(a) through I(k) were tested as follows.

-   -   Dilution of test compounds    -   Stock 10⁻³ M

Concentration From (μL) DMEM + (μL) Concentration (final) previous step1% BSA (actual) 10-5 M 10 115 8 * 10-5 M 10-6 M 12.5 112.5 8 * 10-6 M10-7 M 12.5 112.5 8 * 10-7 M 10-8 M 12.5 112.5 8 * 10-8 M

Compounds of Formula I(a), I(c), I(e), I(g), I(i) and I(k) showedsignificantly greater inhibition of CYP24 than ketoconazole. A graphshowing inhibition of CYP24 activity by compounds I(a) and I(c)(indicated as BH1625(NOH)-TB-2 (CTA062) and BH-1625(NOMe)-TB-2-(CTA065)respectively) compared to ketoconazole is shown in FIG. 14.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications, including Genbankdatabase entries, are herein incorporated by reference in their entiretyto the same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

1. A cell line that stably expresses an active CYP24 polypeptide,comprising a recombinant CYP24 nucleic acid molecule that is operablylinked to a promoter to enable expression thereof, wherein therecombinant CYP24 nucleic acid molecule comprises a nucleic acid havingat least 98% identity to SEQ ID NO: 1 that encodes the active CYP24polypeptide that catalyzes hydroxylation of Vitamin D metabolites. 2.The cell line of claim 1, wherein the cell is a mammalian cell or aninsect cell.
 3. The cell line of claim 1, wherein the cell lineexpresses adrenodoxin (ADX) and adrenodoxin reductase (ADR).
 4. The cellline of claim 1, wherein the cell is a V79 cell or a Sf9 cell.
 5. Thecell line of claim 1, wherein the CYP24 nucleic acid is HPK-1A ras cellCYP24 as shown in SEQ ID NO:
 1. 6. The cell line of claim 1 wherein therecombinant CYP24 nucleic acid molecule is in a vector comprising theCYP24 nucleic acid molecule.
 7. The cell line of claim 6 wherein thevector is pFastBac1, pcDNA3.1 or pcDNA3.1-Hygro(+).
 8. A cell culturethat stably expresses CYP24 polypeptide comprising cells of the cellline of claim
 1. 9. A cell culture comprising cells that stably expressactive CYP24 polypeptide, the cells comprising a recombinant CYP24nucleic acid molecule that is operably linked to a promoter to enableexpression thereof, in a medium capable of sustaining growth andreplication of the cells, wherein the recombinant CYP24 nucleic acidmolecule comprises a nucleic acid having at least 98% identity to SEQ IDNO: 1 that encodes the active CYP24 polypeptide that catalyzeshydroxylation of Vitamin D metabolites.
 10. The cell culture of claim 9,wherein the cells further comprise an antibiotic resistance gene and thecell culture includes an antibiotic encoded by the antibiotic resistancegene.